Nanomechanics controls neuronal precursors adhesion and differentiation

The ability to control the differentiation of stem cells into specific neuronal types has a tremendous potential for the treatment of neurodegenerative diseases. In vitro neuronal differentiation can be guided by the interplay of biochemical and biophysical cues. Different strategies to increase the differentiation yield have been proposed, focusing everything on substrate topography, or, alternatively on substrate stiffness. Both strategies demonstrated an improvement of the cellular response. However it was often impossible to separate the topographical and the mechanical contributions. Here we investigate the role of the mechanical properties of nanostructured substrates, aiming at understanding the ultimate parameters which govern the stem cell differentiation. To this purpose a set of different substrates with controlled stiffness and with or without nanopatterning are used for stem cell differentiation. Our results show that the neuronal differentiation yield depends mainly on the substrate mechanical properties while the geometry plays a minor role. In particular nanostructured and flat polydimethylsiloxane (PDMS) substrates with comparable stiffness show the same neuronal yield. The improvement in the differentiation yield obtained through surface nanopatterning in the submicrometer scale could be explained as a consequence of a substrate softening effect. Finally we investigate by single cell force spectroscopy the neuronal precursor adhesion on the substrate immediately after seeding, as a possible critical step governing the neuronal differentiation efficiency. We observed that neuronal precursor adhesion depends on substrate stiffness but not on surface structure, and in particular it is higher on softer substrates. Our results suggest that cell–substrate adhesion forces and mechanical response are the key parameters to be considered for substrate design in neuronal regenerative medicine. Biotechnol. Bioeng. 2013; 110: 2301–2310. © 2013 Wiley Periodicals, Inc.     

Nanomechanics of Diaminopurine-Substituted DNA

2,6-diaminopurine (DAP) is a nucleobase analog of adenine. When incorporated into double-stranded DNA (dsDNA), it forms three hydrogen bonds with thymine. Rare in nature, DAP substitution alters the physical characteristics of a DNA molecule without sacrificing sequence specificity. Here, we show that in addition to stabilizing double-strand hybridization, DAP substitution also changes the mechanical and conformational properties of dsDNA. Thermal melting experiments reveal that DAP substitution raises melting temperatures without diminishing sequence-dependent effects. Using a combination of atomic force microscopy (AFM), magnetic tweezer (MT) nanomechanical assays, and circular dichroism spectroscopy, we demonstrate that DAP substitution increases the flexural rigidity of dsDNA yet also facilitates conformational shifts, which manifest as changes in molecule length. DAP substitution increases both the static and dynamic persistence length of DNA (measured by AFM and MT, respectively). In the static case (AFM), in which tension is not applied to the molecule, the contour length of DAP-DNA appears shorter than wild-type (WT)-DNA; under tension (MT), they have similar dynamic contour lengths. At tensions above 60 pN, WT-DNA undergoes characteristic overstretching because of strand separation (tension-induced melting) and spontaneous adoption of a conformation termed S-DNA. Cyclic overstretching and relaxation of WT-DNA at near-zero loading rates typically yields hysteresis, indicative of tension-induced melting; conversely, cyclic stretching of DAP-DNA showed little or no hysteresis, consistent with the adoption of the S-form, similar to what has been reported for GC-rich sequences. However, DAP-DNA overstretching is distinct from GC-rich overstretching in that it happens at a significantly lower tension. In physiological salt conditions, evenly mixed AT/GC DNA typically overstretches around 60 pN. GC-rich sequences overstretch at similar if not slightly higher tensions. Here, we show that DAP-DNA overstretches at 52 pN. In summary, DAP substitution decreases the overall stability of the B-form double helix, biasing toward non-B-form DNA helix conformations at zero tension and facilitating the B-to-S transition at high tension.     

Nanomechanics of vascular endothelium

The mechanical characteristics of endothelial cells reveal four distinct compartments, namely glycocalyx, cell cortex, cytoplasm and nucleus. There is accumulating evidence that endothelial nanomechanics of these individual compartments control vascular physiology. Depending on protein composition, filament formation and interaction with cross-linker proteins, these four compartments determine endothelial stiffness. Structural organization and mechanical properties directly influence physiological processes such as endothelial barrier function, nitric oxide release and gene expression. This review will focus on endothelial nanomechanics and its impact on vascular function.     

Nanomechanics of opposing glycosaminoglycan macromolecules

In this study, the net intermolecular interaction force between a chondroitin sulfate glycosaminoglycan (GAG)-functionalized probe tip and an opposing GAG-functionalized planar substrate was measured as a function of probe tip-substrate separation distance in aqueous electrolyte solutions using the technique of high resolution force spectroscopy. A range of GAG grafting densities as near as possible to native cartilage was used. A long-range repulsive force between GAGs on the probe tip and substrate was observed, which increased nonlinearly with decreasing separation distance between probe tip and substrate. Data obtained in 0.1 M NaCl was well predicted by a recently developed Poisson-Boltzmann-based theoretical model that describes normal electrostatic double layer interaction forces between two opposing surfaces of end-grafted, cylindrical rods of constant volume charge density and finite length, which interdigitate upon compression. Based on these results, the nanomechanical data and interdigitated rod model were used together to estimate the electrostatic component of the equilibrium modulus of cartilage tissue, which was then compared to that of normal adult human ankle cartilage measured in uniaxial confined compression.     

Nanomechanics of negatively supercoiled diaminopurine-substituted DNA

Single molecule experiments have demonstrated a progressive transition from a B- to an L-form helix as DNA is gently stretched and progressively unwound. The particular sequence of a DNA segment defines both base stacking and hydrogen bonding that affect the partitioning and conformations of the two phases. Naturally or artificially modified bases alter H-bonds and base stacking and DNA with diaminopurine (DAP) replacing adenine was synthesized to produce linear fragments with triply hydrogen-bonded DAP:T base pairs. Both unmodified and DAP-substituted DNA transitioned from a B- to an L-helix under physiological conditions of mild tension and unwinding. This transition avoids writhing and the ease of this transition may prevent cumbersome topological rearrangements in genomic DNA that would require topoisomerase activity to resolve. L-DNA displayed about tenfold lower persistence length than B-DNA. However, left-handed DAP-substituted DNA was twice as stiff as unmodified L-DNA. Unmodified DNA and DAP-substituted DNA have very distinct mechanical characteristics at physiological levels of negative supercoiling and tension.     

Adenomatous polyposis coli proteins and cell adhesion

Adenomatous polyposis coli (APC) is an important tumour suppressor in the mammalian intestinal epithelium. It binds to beta-catenin and its role as a tumour suppressor depends predominantly on its ability to downregulate soluble beta-catenin, a key effector of the Wnt signalling pathway. However, epithelial cells have a distinct subcellular pool of beta-catenin, or Drosophila Armadillo, which functions as a structural component of adherens junctions. Notably, APC proteins can be associated with these adherens junctions, and recent evidence points to a role for APC in cellular adhesion. Thus, APC--like beta-catenin/Armadillo--may have a dual role in Wnt signal transduction and in cellular adhesion, which could be relevant to its activity as a tumour suppressor.     

Extracellular RGD-binding proteins modulate cell adhesion.

Cell/fibronectin adhesion in extracellular matrices is partly mediated by integrin receptor recognition of RGD domains in fibronectin. Since blood contains significant levels of soluble fibronectin we have now investigated the occurrence of extracellular RGD-binding proteins. Attachment assays indicate that extracellular RGD-binding proteins prevent cell adhesion, suggesting their potential as novel secreted modulators of blood-borne cell adhesive interactions. These extracellular RGD-binding proteins also showed electrophoretic changes with reducing agents, suggestive of intrachain disulphide bonds, like those found in RGD-binding integrins. However, they differed from the latter in their electrophoretic profile, which was greatly dependent on the presence of protease inhibitors. Plasma from tumor-bearing mice showed a greater proportion of fast-migrating RGD-binding species under reducing condition compared to similarly treated normal plasma, suggesting that tumor development is associated with a partial degradation of extracellular RGD-binding proteins.     

Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in solution, has shown considerable potential in biochemical sensing applications with unprecedented sensitivity and specificity^1-5. The drug-target interactions generate surface stress, causing the cantilever to bend, and the signal can be analyzed optically when it is illuminated by a laser. The change in surface stress measured with nano-scale precision allows disruptions of the biomechanics of model bacterial cell wall targets to be tracked in real time. Despite offering considerable advantages, multiple cantilever sensor arrays have never been applied in quantifying drug-target binding interactions.Here, we report on the use of silicon multiple cantilever arrays coated with alkanethiol self-assembled monolayers mimicking bacterial cell wall matrix to quantitatively study antibiotic binding interactions. To understand the impact of vancomycin on the mechanics of bacterial cell wall structures^1,6,7. We developed a new model¹ which proposes that cantilever bending can be described by two independent factors; i) namely a chemical factor, which is given by a classical Langmuir adsorption isotherm, from which we calculate the thermodynamic equilibrium dissociation constant (K_d) and ii) a geometrical factor, essentially a measure of how bacterial peptide receptors are distributed on the cantilever surface. The surface distribution of peptide receptors (p) is used to investigate the dependence of geometry and ligand loading. It is shown that a threshold value of p ~10% is critical to sensing applications. Below which there is no detectable bending signal while above this value, the bending signal increases almost linearly, revealing that stress is a product of a local chemical binding factor and a geometrical factor combined by the mechanical connectivity of reacted regions and provides a new paradigm for design of powerful agents to combat superbug infections.     

Binding of plasminogen to Escherichia coli adhesion proteins

Immobilization of plasminogen via its lysine-binding sites is regarded as a prerequisite for its activation and function in fibrinolysis and pericellular proteolysis. In the present study, the interaction of plasminogen with fimbriae found on Escherichia coli strains causing invasive human infections was studied. Plasminogen displayed concentration-dependent and saturable binding to immobilized type 1 fimbriae and, several fold lower binding to P and S fimbriae. The binding to fimbriae was effectively inhibited by -aminocaproic acid indicating that it was mediated by the lysine-binding sites of plasminogen. Binding studies with mutated fimbriae and inhibition tests indicated that the interaction was not dependent on the lectin subunit of the fimbriae. These results indicate the existence of a novel type of host-microbe interaction which may be important in the invasion by bacteria of host tissues.     

Nanomechanics of the Endothelial Glycocalyx in Experimental Sepsis

The endothelial glycocalyx (eGC), a carbohydrate-rich layer lining the luminal side of the endothelium, regulates vascular adhesiveness and permeability. Although central to the pathophysiology of vascular barrier dysfunction in sepsis, glycocalyx damage has been generally understudied, in part because of the aberrancy of in vitro preparations and its degradation during tissue handling. The aim of this study was to analyze inflammation-induced damage of the eGC on living endothelial cells by atomic-force microscopy (AFM) nanoindentation technique. AFM revealed the existence of a mature eGC on the luminal endothelial surface of freshly isolated rodent aorta preparations ex vivo, as well as on cultured human pulmonary microvascular endothelial cells (HPMEC) in vitro. AFM detected a marked reduction in glycocalyx thickness (266 ± 12 vs. 137 ± 17 nm, P<0.0001) and stiffness (0.34 ± 0.03 vs. 0.21 ± 0.01 pN/mn, P<0.0001) in septic mice (1 mg E. coli lipopolysaccharides (LPS)/kg BW i.p.) compared to controls. Corresponding in vitro experiments revealed that sepsis-associated mediators, such as thrombin, LPS or Tumor Necrosis Factor-α alone were sufficient to rapidly decrease eGC thickness (-50%, all P<0.0001) and stiffness (-20% P<0.0001) on HPMEC. In summary, AFM nanoindentation is a promising novel approach to uncover mechanisms involved in deterioration and refurbishment of the eGC in sepsis.     

Synergistic control of cellular adhesion by transmembrane 9 proteins

The transmembrane 9 (TM9) family of proteins contains numerous members in eukaryotes. Although their function remains essentially unknown in higher eukaryotes, the Dictyostelium discoideum Phg1a TM9 protein was recently reported to be essential for cellular adhesion and phagocytosis. Herein, the function of Phg1a and of a new divergent member of the TM9 family called Phg1b was further investigated in D. discoideum. The phenotypes of PHG1a, PHG1b, and PHG1a/PHG1b double knockout cells revealed that Phg1a and Phg1b proteins play a synergistic but not redundant role in cellular adhesion, phagocytosis, growth, and development. Complementation analysis supports a synergistic regulatory function rather than a receptor role for Phg1a and Phg1b proteins. Together, these results suggest that Phg1 proteins act as regulators of cellular adhesion, possibly by controlling the intracellular transport in the endocytic pathway and the composition of the cell surface.     

Increased adhesion of lymphoid cells to glycated proteins.

BACKGROUND AND AIMS: The advanced glycation end-products are involved in the pathogenesis of vascular damages and other clinical complications in diabetic patients. The aim of this study was to investigate the adhesion of lymphoid cells to nonenzymatically glycated proteins in comparison with the unmodified substances. METHODS: Two cell lines (monocyte-macrophage line U937 and the T-cell line Jurkat) were used throughout the experiments. The cells were left to adhere to nonenzymatically glycated and native proteins coated on a 96-well flat-bottom plates and the cellular adhesion was registered as absorption at 550 nm following the method described by Ivanov and Kyurkchiev [G. Ivanov, S. Kyurkchiev, Effect of advanced glycosylation end-products on the activity of integrins expressed on U937 cells, Hum. Immunol. 59 (1998) 325-330.]. RESULTS: It was found that the monocytes had increased adhesion to nonenzymatically glycated proteins such as collagen, fibronectin and bovine serum albumin, whereas the T-cells had increased adhesion to the glycated collagen and bovine serum albumin but reduced adhesion to advanced glycated fibronectin. Experiments with different stimulating agents showed that phorbol-myriastate, acetate (A550 = 0.672 +/- 0.068, S.E.M., n = 40), glucose (A550 = 0.593 +/- 0.051, S.E.M., n = 40) and TNF-alpha (A550 = 0.580 +/- 0.042, S.E.M., n = 40) increased the adhesion of U937 cells to advanced glycated bovine serum albumin in comparison with the adhesion of the untreated cells (A550 = 0.260 +/- 0.046, S.E.M., n = 40). This is probably due to an upregulation of the expression or the activity of the receptors for the advanced glycation end-products. CONCLUSION: Based on the results obtained it is concluded that the receptors for nonenzymatically glycated proteins expressed on the surface of lymphoid cells could act also as cell adhesion molecules.     

Csk enhances insulin-stimulated dephosphorylation of focal adhesion proteins.

Insulin has pleiotropic effects on the regulation of cell physiology through binding to its receptor. The wide variety of tyrosine phosphorylation motifs of insulin receptor substrate 1 (IRS-1), a substrate for the activated insulin receptor tyrosine kinase, may account for the multiple functions of insulin. Recent studies have shown that activation of the insulin receptor leads to the regulation of focal adhesion proteins, such as a dephosphorylation of focal adhesion kinase (pp125FAK). We show here that C-terminal Src kinase (Csk), which phosphorylates C-terminal tyrosine residues of Src family protein tyrosine kinases and suppresses their kinase activities, is involved in this insulin-stimulated dephosphorylation of focal adhesion proteins. We demonstrated that the overexpression of Csk enhanced and prolonged the insulin-induced dephosphorylation of pp125FAK. Another focal adhesion protein, paxillin, was also dephosphorylated upon insulin stimulation, and a kinase-negative mutant of Csk was able to inhibit the insulin-induced dephosphorylation of pp125FAK and paxillin. Although we have shown that the Csk Src homology 2 domain can bind to several tyrosine-phosphorylated proteins, including pp125FAK and paxillin, a majority of protein which bound to Csk was IRS-1 when cells were stimulated by insulin. Our data also indicated that tyrosine phosphorylation levels of IRS-1 appear to be paralleled by the dephosphorylation of the focal adhesion proteins. We therefore propose that the kinase activity of Csk, through the insulin-induced complex formation of Csk with IRS-1, is involved in insulin's regulation of the phosphorylation levels of the focal adhesion proteins, possibly through inactivation of the kinase activity of c-Src family kinases.     

The collagen family members as cell adhesion proteins

The collagen family of extracellular matrix proteins has played a fundamental role in the evolution of multicellular animals. At the present, 28 triple helical proteins have been named as collagens and they can be divided into several subgroups based on their structural and functional properties. In tissues, the cells are anchored to collagenous structures. Often the interaction is indirect and mediated by matrix glycoproteins, but cells also express receptors, which have the ability to directly bind to the triple helical domains in collagens. Some receptors bind to sites that are abundant in all collagens. However, increasing evidence indicates that the coevolution of collagens and cell adhesion mechanisms has given rise to receptors that bind to specific motifs in collagens. These receptors may also recognize the different members of the large collagen family in a selective manner. This review summarizes the present knowledge about the properties of collagen subtypes as cell adhesion proteins.     

Nanomechanics of Native Thick Filaments from Indirect Flight Muscles

During flight, the wings of Drosophila melanogaster beat nearly 200 times per second. The indirect flight muscle fibers that power this movement have evolved to resist the repetitive mechanical stress that results from the 5-ms wing beat cycle at a strain amplitude of 3.5%. In order to understand how this is achieved at the sarcomere level, we have analyzed the mechanical properties of native thick filaments isolated from indirect flight muscle. Single filaments adsorbed onto a solid support were manipulated in physiological buffer using an atomic force microscope. Images taken after the manipulation revealed that segments were stretched, on average, to 150%, with a maximum at 385% extension. The lateral-force-versus-displacement curve associated with each manipulation contained information about the bending and tensile properties of each filament. The bending process was dominated by shearing between myosin dimers and yielded a shear modulus between 3 and 13 MPa. Maximum tension along the stretched filaments was observed at ∼ 200% extension and varied between 8 and 17 nN. Based on current models of thick filament structure, these variations can be attributed to cross-links between myosin dimers distributed along the filament.     

Comparing mechano-transduction in fibroblasts deficient of focal adhesion proteins

Mechano-transduction was studied in wildtype and focal adhesion (FA) protein-deficient mouse embryonic fibroblasts (MEFs). Using a cell stretcher, we determined the effect of stretch on cell morphology, apoptosis, and phosphorylation of ERK^1/2. After 20% cyclic, uniaxial stretch, FA-deficient MEFs showed morphological changes and levels of apoptosis of the order: focal adhesion kinase > p130Cas > vinculin compared to wildtype cells. ERK^1/2 phosphorylation peaked in wildtype cells at around 10 min, and in all FA-deficient cells at around 5 min. The relative change in strain energy of FA-deficient cells compared to wildtype cells was of the order: vinculin > FAK > p130Cas. Taken together, FAK and p130Cas are more important in the stretch-mediated downstream signaling and cell survival pathway, while vinculin is more critical in maintaining cell contractility.     

Nanomechanics of G-quadruplexes within the promoter of the KIT oncogene

G-quadruplexes (G4s) are tetrahelical DNA structures stabilized by four guanines paired via Hoogsteen hydrogen bonds into quartets. While their presence within eukaryotic DNA is known to play a key role in regulatory processes, their functional mechanisms are still under investigation. In the present work, we analysed the nanomechanical properties of three G4s present within the promoter of the KIT proto-oncogene from a single-molecule point of view through the use of magnetic tweezers (MTs). The study of DNA extension fluctuations under negative supercoiling allowed us to identify a characteristic fingerprint of G4 folding. We further analysed the energetic contribution of G4 to the double-strand denaturation process in the presence of negative supercoiling, and we observed a reduction in the energy required for strands separation.